Development of a Medical Test.
The probability of curing a disease (e.g. a cancer disease) is many times predominantly dependent from an early as possible detection of the disease. It is also often advantageous to detect a predisposition for a disease or if for example the disease is already advanced to make an estimation for the most promising treatment for the disease. Such an early as possible detection, prediction or estimation reduces the costs for direct and associated medical treatment. It ensures also a higher quality of life for the affected patient.
This leads to the situation that a lot of samples derived from individuals with a suspected disease have to be tested, the majority may not be affected by the disease. Or, in case of patients with a diagnosed disease, a lot of samples have to be tested, and only a small percentage will respond to a certain treatment. Usually the majority of said tests is performed in so-called reference laboratories. Because of the huge number of samples which are processed in reference laboratories, the actual test and the corresponding workflow of processing the sample underlie the following requirements: combinable with methods of carry over prevention; low demands on laboratory equipment; low handling effort; capable of being automated by means of robotics or manually; capable of being standardized; realizable in plate scale; high yield of DNA in order to reduce the amount of sample and to increase the sensitivity and specificity; high sensitivity; high specificity; low costs; DNA free of interfering compounds such as but not limited to proteins, RNA, nucleotides or disturbing chemical reagents; high reproducibility and high reliability.
Furthermore, in general, it is desirable that a test should have a high as possible sensitivity, a high as possible specificity and a high as possible accuracy. Sensitivity is a measure of a test's ability to correctly detect the target disease in an individual being tested. A test having poor sensitivity produces a high rate of false negatives, i.e., Individuals who have the disease but are falsely identified as being free of that particular disease. The potential danger of a false negative is that the diseased individual will remain undiagnosed and untreated for some period of time, during which the disease may progress to a later stage wherein treatments, if any, may be less effective. Mathematical it can be described as: Sensitivity=TP/(TP+FN). Thereby TP represents a true positive result and FN a false negative result. A true positive result means that the test is positive and the condition is present while a false negative result is where the test is negative but the condition is not present.
Specificity, on the other hand, is a measure of a test's ability to identify accurately patients who are free of the disease state. A test having poor specificity produces a high rate of false positives, i.e., individuals who are falsely identified as having the disease. A drawback of false positives is that they force patients to undergo unnecessary medical procedures or treatments with their attendant risks, emotional and financial stresses, and which could have adverse effects on the patient's health. A feature of diseases which makes it difficult to develop diagnostic tests with high specificity is that disease mechanisms, particularly in cancer, often involve a plurality of genes and proteins. Additionally, certain proteins may be elevated for reasons unrelated to a disease state. Mathematical specificity can be described as: Specificity=TN/(FP+TN). Thereby TN represents a true negative result and FP a false positive result. A true negative result is where the test is negative and the condition is not present. A false positive result is where the test is positive but the condition is not present.
Starting Material for a Test.
It is advantageous for a test with regard to cost reduction and to a high quality of life of the patient that it can be performed non-invasively. If this is not possible, it is desirably to perform it by invasive means which affect as less as possible the patient, which are easy to perform, which cause low costs or combinations thereof. Because of that, remote samples such as but not limited to blood, sputum, stool or body fluids are the starting material of choice for a test.
However, the use of remote samples is quite limited by the low amount of DNA, in particular by the low amount of DNA which originates by the diseased cell or tissue. Therefore the workflow from the sample collecting to the start of the test has to be characterized by high yields of DNA.
Furthermore, the DNA of interest might be partially degraded in a remote sample. This depends on the type of the remote sample and also on the way of collecting and handling the remote sample. A fragmentation of DNA in remote sample down to a fragment size of 100 bp and under it is possible. Therefore a workflow from collecting a sample to the start of a test should ensure that small DNA fragments as well as large DNA fragments are provided and that the DNA does not get further fragmented. Numerous documents exist which address these problems. Exemplary only the following are cited herein: Diehl F., et al. (2005) PNAS 102(45), 16368-16373; and Li J., et al. (2006) Journal of Molecular Diagnostics, 8(1), 22-30.
Methylation Analysis.
As revealed in recent years, one of the most powerful and promising approaches for detecting a disease, the pre-disposition for a disease or for estimating a probable response with respect to a certain disease treatment is the methylation analysis of the patient's genomic DNA.
Many diseases, in particular cancer diseases, are accompanied by modified gene expression. This may be a mutation of the genes themselves, which leads to an expression of modified proteins or to an inhibition or over-expression of the proteins or enzymes. A modulation of the expression may however also occur by epigenetic modifications, in particular by changes in the DNA methylation pattern. Such epigenetic modifications do not affect the actual DNA coding sequence. It has been found that DNA methylation processes have substantial implications for health, and it seems to be clear that knowledge about methylation processes and modifications of the methyl metabolism and DNA methylation are essential for understanding diseases, for the prophylaxis, diagnosis and therapy of diseases.
The precise control of genes, which represent a small part only of the complete genome of mammals, involves regulation in consideration of the fact that the main part of the DNA in the genome is not coding. The presence of such ‘trunk’ DNA containing introns, repetitive elements and potentially actively transposable elements, requires effective mechanisms for their durable suppression (silencing). Apparently, the methylation of cytosine by S-adenosylmethionine (SAM) dependent DNA methyl transferases, which form 5-methylcytosine, represents such a mechanism for the modification of DNA-protein interactions. Genes can be transcribed by methylation-free promoters, even when adjacent transcribed or not-transcribed regions are widely methylated. This permits the use and regulation of promoters of functional genes, whereas the trunk DNA including the transposable elements is suppressed. Methylation also takes place for the long-term suppression of X-linked genes and may lead to either a reduction or an increase of the degree of transcription, depending on where the methylation in the transcription units occurs.
Nearly the complete natural DNA methylation in mammals is restricted to cytosine-guanine (CpG) dinucleotide palindrome sequences, which are controlled by DNA methyl transferases. CpG dinucleotides are about 1 to 2% of all dinucleotides and are concentrated in CpG islands. According to an art-recognized definition, a region is considered as a CpG island when the C+G content over 200 bp is at least 50% and the percentage of the observed CG dinucleotides in comparison to the expected CG dinucleotides is larger than 0.6 (Gardiner-Garden, M., Frommer, M. (1987) J. Mol. Biol. 196, 261-282). Typically, CpG islands have at least 4 CpG dinucleotides in a sequence of a length of 100 bp.
CpG islands located in promotor regions frequently have a regulatory function for the expression of the corresponding gene. For example, in case the CpG island is hypomethylated, the gene can be expressed. On the other hand, hypermethylation frequently leads to a suppression of the expression. Normally tumor suppressor genes are hypomethylated. But if they become hypermethylated, their expression becomes suppressed. This is observed many times in tumor tissues. By contrast, oncogenes are hypermethylated in healthy tissue, whereas they are hypomethylated in many times in tumor tissues.
The methylation of cytosine has the effect that the binding of proteins is normally prohibited which regulate the transcription of genes. This leads to an alteration of the expression of the gene. Relating to cancer, the expression of genes regulating cell division are thereby altered, for example, the expression of an apoptotic gene Is down regulated, while the expression of an oncogene is up regulated. Additionally, hypermethylation may have a long term influence on regulation. Proteins, which deacetylate histones, are able to bind via their 5-methylcytosine binding domain to the DNA when the cytosines get methylated. This results in a deacetylation of the histones, which itself leads to a tighter package of the DNA. Because of that, regulatory proteins are not precluded from binding to the DNA.
The efficient detection of DNA methylation patterns consequently is an important tool for developing new approaches to understand diseases, for the prevention, diagnosis and treatment of diseases and for the screening for disease associated targets.
Background of the Septin 9 Gene.
The human Septin 9 gene (also known as MLL septin-like fusion protein, MLL septin-like fusion protein MSF-A, Sipa, Eseptin, Msf, septin-like protein Ovarian/Breast septin (Ov/Br septin) and Septin D1) is located on chromosome 17q25 within contig AC068594.15.1.168501 and is a member of the Septin gene family. The Septin 9 gene is known to comprise four transcript variants, the Septin 9 variants and the Q9HC74 variants (which are truncated versions of the Septin 9 transcripts). The Septin 9 and Q9HC74 transcripts comprise each a CpG rich promotor region, respectively. It has been postulated that members of the Septin gene family are associated with multiple cellular functions ranging from vesicle transport to cytokinesis. Disruption of the action of Septin 9 results in incomplete cell division, see Surka, M. C., Tsang, C. W., and Trimble, W. S. Mol Biol Cell, 13: 3532-45 (2002). Septin 9 and other proteins have been shown to be fusion partners of the protooncogene MLL suggesting a role in tumorogenesis, see Osaka, M, Rowley, J. D. and Zeleznik-Le, N. J. PNAS, 96:6428-6433 (1999). Burrows et al. reported an in depth study of expression of the multiple isoforms of the Septin 9 gene in ovarian cancer and showed tissue specific expression of various transcripts, see Burrows, J. F., Chanduloy, et al. S. E. H. Journal of Pathology, 201:581-588 (2003). A recent study of over 7000 normal and tumor tissues indicates that there is consistent over-expression of Septin 9 isoforms in a number of tumor tissues, see Scott, M., Hyland, P. L., et al. Oncogene, 24: 4688-4700 (2005). The authors speculate that the gene is likely a type II cancer gene where changes in RNA transcript processing control regulation of different protein products, and the levels of these altered protein isoforms may provide answers to the gene's role in malignancy.
State of the Art.
As the closest prior art, the following documents may be considered:
Utting M., et al. (2002) Clinical Cancer Research 8, 35-40, This study indicates that microsatellite marker analysis using free-floating DNA of urine or blood could be relevant for diagnosis and screening of bladder cancer. The sample providing as well as the providing of DNA from the samples is carried out according to standard procedures.
Wong I. H. N., et al. (2003) Clinical Cancer Research 9, 047-1052 describe a new method named RTQ-MSP which is a combination of MSP (methylation sensitive PCR) and real-time PCR. The authors demonstrate that a detection of a particular tumor-derived DNA sequence in plasma, serum and blood cells of already diagnosed hepatocellular carcinoma patients is possible.
U.S. Pat. No. 6,927,028 teaches a method for differentiating DNA species originating form cells of different individuals in biological samples by means of methylation specific PCR. The sample providing as well as the providing of DNA from the samples is carried out according to standard procedures.
Lecomte T., et al. (2002) Int. J. Cancer 100, 542-548 tested free-circulating DNA derived from plasma of colorectal cancer patients for the presence of KRAS2 mutations, for p16 gene promoter methylation, or both. The authors suggest, patients with free-circulating tumor-associated DNA in the blood have a lower probability of a 2-year recurrence-free survival than patients for who no free-circulating tumor-associated DNA in the blood is detected.
WO 2006/039563 relates to compositions and methods for providing DNA fragments from an archived sample like paraffin-embedded and/or formalin-fixed tissue biopsies. It discloses methods wherein high yields of DNA are isolated as well as a substantial portion of the DNA consists of long DNA fragments, and where the isolated genomic DNA is free of associated or cross-linked contaminants like proteins, peptides, amino acids or RNA. The methods are facile, cost-effective, and are characterized by high reproducibility and reliability. Particularly, methods are disclosed for providing DNA fragments derived from an archived sample, wherein the yield of DNA before an amplification step is at least 20%, and amplicons up to a length of about 1,000 base pairs are amplifiable.
WO 2006/113770 discloses compositions and methods for providing DNA fragments from a remote sample. Accordingly, DNA is isolated from the remote sample, and the isolated DNA is treated in a way which allows differentiation of methylated and unmethylated cytosine. Additional, particular embodiments provide compositions and methods for methylation analysis of DNA derived from a remote sample. WO 2006/113770 discloses compositions and methods of whole genome amplification of bisulfite treated DNA.
WO 2006/113466, EP 1721992 and US 20060286576 provide methods, nucleic acids and kits for detecting, or for detecting and distinguishing between or among liver cell proliferative disorders or for detecting, or for detecting and distinguishing between or among colorectal cell proliferative disorders based on the Septin 9 gene and its methylation. Particular aspects disclose and provide genomic sequences of the Septin 9 gene, the methylation patterns of which have substantial utility for the improved detection of and differentiation between said class of disorders, thereby enabling the improved diagnosis and treatment of patients.
However, non of the methods of the state of the art is suitable for the application in reference laboratories. Therefore a pronounced need in the art exists for compositions and methods for providing DNA for methylation analysis that is suitable for the use in reference laboratories. Therefore, in addition, a pronounced need in the art exists for compositions and methods for the analysis of the methylation of the Septin 9 gene that is applicable in reference laboratories.